A large percentage of investigation in the biochemical arts are directed to studies involving nucleic acids, particularly deoxyribonucleic acid, or DNA. DNA is a water-soluble compound, that if left in solution (i.e., a water-based solution), is likely to degrade, through hydrolysis, and so forth. Obviously this frustrates any investigation involving DNA, and so therefore, accurate and reliable study involving DNA requires a method or device to ensure the integrity of DNA. To facilitate the study of DNA, it is often desirable to affix or immobilize the DNA on a solid surface, such as a smooth sheet of glass. Fixed in place in this manner, the DNA can be readily manipulated (i.e., reacted with other substances). If DNA is envisioned as a long strand, then immobilizing DNA means fixing one end of the strand to the solid support so that the remainder of the strand is unmodified and free to undergo further reaction depending upon the particular study. Indeed, this is a widely used method to conduct laboratory studies involving DNA.
Perhaps the major problem associated with immobilizing DNA on a solid support is exactly how to do it without altering the DNA (other than that relatively small portion that is actually bound to the solid support). This is a very difficult problem because whatever solid support is used must be essentially inert. That is, it must not react with the DNA, other than simply to immobilize it upon the solid support. Glass is a particularly suitable solid support, because it is inexpensive, and highly inert. At present, the current orthodoxy is that the solid support (e.g., a glass surface) must first be primed or derivatized so that it can bind (one end of) the DNA to the surface. Numerous techniques exist to do this.
Unfortunately, derivatizing the other wise inert surface of glass creates problems which could confound the results of the laboratory study involving DNA. One problem is that derivatization activates and sometime creates a new positive electrostatic charge on the glass surface. Since DNA is (net) negatively charged, other DNA (or DNA used later in the study but not deliberately affixed to the glass surface) is prone to stick (by non-specific electrostatic attraction) to the glass surface. In other words, DNA “probes” which are single (rather than double) strands of DNA are often contacted with an array of DNA single strands affixed to a solid support. Since the probe has a known nucleotide sequence and since a particular single strand of DNA will bind preferentially to a complementary strand, the particular immobilized strand to which the probe reacts reveals the nucleotide sequence of the previously unknown immobilized strand. Yet simple experiments of this type (probe studies) are severely confounded by electrostatic sticking of the probe to the derivatized (hence electrostatically charged) glass surface. For instance, the probe is often radiolabeled so that is presence can be detected by an ordinary radiation detector. Thus, the location of the probe on the glass surface, as evidenced by the detector, reveals the chemical identity or sequence of the immobilized DNA strand at that particular location on the glass surface (which is known and designated in advance). Yet the radiation detector is unable to distinguish between probe that is chemically bound to a complementary strand of DNA affixed to the solid support, and probe that is simply electrostatically stuck to the glass surface (but not to a DNA strand).
Second, derivatized surfaces result in what shall be known as “spreading.” Spreading occurs because the solid support surface becomes hydrophilic upon derivatization. As a result, when the DNA (desired to be immobilized upon the solid support) is contacted with the surface of the solid support, it spreads, rather than remaining in a discrete “spot,” which it should ideally do, since whether the radioactive probe is detected in one spot or another determines whether the scientist infers that the probe reacted with this or that immobilized DNA. Spreading is a major constraint on array density (i.e., the number of different nucleic acid samples that can be arranged on a single solid support). Hence, any means to curtail spreading, and so increase array density, is highly desirable.
One very common substance used to prepare a glass surface to receive a nucleic acid sample is poly-L-lysine. See, e.g., DeRisi, et al., Use of a cDNA Microarray to Analyze Gene Expression Pattern in Human Cancer, 14 Nature Genetics 457 (1996),; Shalon et al. in a A DNA Microarray System for Analyzing Complex DNA samples Using Two-Color Fluorescent Probe Hybridization, 6 Genome Res. 639 (1996); and Schena, et al., Quantitative Monitoring of Gene Expression Patterns With a Complementary DNA Microarray, 270 Science 467 (1995). Other types of pre-derivatized glass supports are commercially available (e.g., silylated microscope slides). See, e.g., Schena, et al., Parallel Human Genome Analysis: Microarray-Based Expression Monitoring of 1000 Genes, 93 P.N.A.S. 10614 (1996).
Numerous other surface coatings have been disclosed. See, e.g., U.S. Pat. No. 5,630,932, assigned to Molecular Imaging Corp., discloses a coating for a probe (platinum) tip for use in scanning tunneling microscopy; numerous means are disclosed for coating the surface, notably, Si(OHC3)CH2I. U.S. Pat. No. 5,610,287, assigned to Molecular Tool, discloses coating a solid support with a salt or cationic detergent to non-covalently bond nucleic acids to the support. U.S. Pat. No. 5,024,933, assigned to Enzo Biochem, discloses coating a solid support with an isolate of naturally occurring mussel adhesive protein. U.S. Pat. No. 4,937,188, assigned to Northeastern University, discloses covalently bonding an enzyme to a solid support via molecular chain which acts as a substrate for the enzyme. U.S. Pat. No. 4,818,681, assigned to Molecular Diagnostics, discloses coating a solid support with a nucleoside phosphate through the heterocyclic moiety of the nucleoside; the nucleic acid is them immobilized upon the solid support by enzymatic coupling. U.S. Pat. No. 4,806,631, assigned to Miles, discloses activating a nylon solid support by partially solvolyzing the amine groups (e.g., by treating with an alkylating group) on the nylon surface.
Another approach to this problem involves derivatizing both the solid support and the nucleic acid sought to be immobilized. See, e.g., U.S. Pat. No. 5,641,630, assigned to Amgen and Abbott, discloses coating a solid support with a complexing agent that binds to an other complexing agent to which the nucleic acid sought to be bound is likewise bound. U.S. Pat. No. 5,554,744, assigned to Hybridon, discloses contacting a solid support with diisopropylcarbodiimide and an acid catalyst and a succinylated nucleoside to immobilize the nucleoside. U.S. Pat. No. 5,514,785, assigned to Becton Dickinson, discloses coating a solid support with, preferably, primary and secondary amines, followed by activation of the nucleic acid using cyanuric chloride. U.S. Pat. No. 5,215,882, assigned to Ortho Diagnostic Systems, discloses modifying the nucleic acid sought to be immobilized with a primary amine or equivalent, followed by reaction of the modified nucleic acid with the solid support (the support must have free aldehyde groups) in the presence of a reducing agent.
Finally, a third approach to the problem of immobilizing nucleic acids to solid support material involves creating a novel solid support. See, e.g., U.S. Pat. Nos. 5,055,429, 5,008,220, 4,963,436, 4,826,790, and 4,826,789, assigned to ECC International, disclose solid support material made from aluminsilicate material.
Due to the aforementioned shortcomings of derivatizing the (entire) glass surface prior to affixing the nucleic acid samples, several methods have been developed which involve synthesizing the nucleic acid samples directly to the solid support. See, e.g., Hacia, et al., Detection of Heterozygous Mutations in BRCA1 Using High Density Oligonucleotide Arrays and Two-Colour Fluorescence Analysis, 14 Nature Genetics 441 (1996); Lockhart, et al., Expression Monitoring by Hybridization to High-Density Oligonucleotide Arrays, 14 Nature Biotechnology 1675 (1996); Maskos and Southern, Oligonucleotide Hybridizations on Glass Supports: a Novel Linker for Oligonucleotide Synthesis and Hybridization Properties of Oligonucleotides Synthesized In Situ, 20 Nucleic Acids Res. 1679 (1992) (and references cited there, particularly 5-11).
To reiterate: at present, the prevailing view in the biochemical arts is that, in order to effectively immobilize nucleic acids onto solid surfaces, the solid support must first be derivatived, or made chemically labile, so that the nucleic acid can then be reacted with solid support. In addition, epoxides are known mutagens; that is, they are known to damage nucleic acids, particularly DNA.
This invention demonstrates that any biological molecule can be modified and affixed to an unmodified solid support. A skilled artisan will recognize the significance of first modifying a molecule to enhance its binding affinity by the appropriate modifications known in the art, thus this modified molecule can be immobilized to an unmodified solid surface generating a fully functional array of molecules for a spectrum of specific applications.